The viability of MM.1S, INA-6, RPMI-8226, MM.1R, Dox-40, ARP-1, and ANBL-6 cells is lowered in a concentration-dependent manner by melflufen (melphalan flufenamide) (0.5-10 μM; 24 hours)[1]. In MM.1S cells, melflufen causes apoptosis [1]. Additionally, melphalan is a strong exosome secretion activator [3].

In a xenograft mouse model, melflufen (3 mg/kg; intravenously administered twice weekly for two weeks) exhibits anti-MM action [1].

Cell viability assay
Cell Types: Multiple myeloma cells: MM.1S, INA-6, RPMI-8226, MM.1R, Dox-40, ARP-1, ANBL-6 Cell
Tested Concentrations: 0.5, 1, 3, 5, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: A significant concentration-dependent decrease in viability was observed for all cell lines.

Animal/Disease Models: CB-17 SCID mouse (human plasmacytoma MM.1S xenograft mouse model) [1]
Doses: 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection; twice a week for two weeks
Experimental Results: Significant Dramatically inhibits MM tumor growth and prolongs mouse survival.

Absorption, Distribution and Excretion
Following an intravenous infusion of 40 mg, the peak plasma concentration (Cmax) of the active metabolite was 432 ng/mL, the time to peak concentration (Tmax) was 4–15 minutes, and the area under the curve (AUC) was 3143 hµg/mL. Elimination pathway data for melphalan-fluoroaniline are unclear. Free melphalan undergoes rapid spontaneous degradation, complicating studies on its elimination pathway. However, it is expected to be primarily excreted via the kidneys. Mean Volume of Distribution (VOD) for melphalan-fluoroaniline is 35 L, while for melphalan it is 76 L. Mean Clearance (MSC) for melphalan-fluoroaniline is 692 L/h, while for melphalan it is 23 L/h. Metabolisms/Metabolites Melphalan-fluoroaniline is metabolized to desethylmelphalan and melphalan. Melphalan can spontaneously hydrolyze to monohydroxymelphalan and dihydroxymelphalan.

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Biological Half-Life
The mean elimination half-life of melphalan is 2.1 minutes, while the mean elimination half-life of melphalan is 70 minutes.

Protein Binding
Data regarding the protein binding of melphalan fluoroaniline are unclear. However, free melphalan binds 60% to albumin, 20% to α-1-acid glycoprotein, and 10% to other proteins in plasma.

[1]. In vitro and in vivo antitumor activity of a novel alkylating agent, melphalan-flufenamide, against multiple myeloma cells. Clin Cancer Res. 2013;19(11):3019-3031.

[2]. A novel alkylating agent Melflufen induces irreversible DNA damage and cytotoxicity in multiple myeloma cells. Br J Haematol. 2016;174(3):397-409.

[3]. Mechanisms associated with biogenesis of exosomes in cancer. Mol Cancer. 2019 Mar 30;18(1):52.

Melphalan fluoroaniline (also known as melphalan or J1) is a prodrug of melphalan. Melphalan fluoroaniline is more readily absorbed by cells than melphalan and is hydrolyzed by aminopeptidase into its active metabolite. In vitro models show that melphalan fluoroaniline is 10 to several hundred times more potent than melphalan. This enhanced potency makes melphalan fluoroaniline a treatment option for patients with relapsed or refractory multiple myeloma who have previously tried at least four lines of therapy. Melphalan fluoroaniline was approved by the FDA on February 26, 2021. However, due to results from the Phase 3 OCEAN trial showing that while melphalan fluoroaniline provided superior progression-free survival compared to the standard treatment regimen of pomalidomide and dexamethasone, it resulted in a decline in overall survival, leading to its withdrawal from the market. Melphalan fluoroaniline is an alkylating agent. Its mechanism of action is alkylation.
Melphalan fluoroaniline is a peptide-drug conjugate composed of a peptide coupled to the alkylating agent melphalan via aminopeptidase targeting, possessing potential antitumor and anti-angiogenic activities. After administration, the highly lipophilic melphalan fluoroaniline can penetrate the cell membrane and enter cells. In aminopeptidase-positive tumor cells, melphalan fluoroaniline is hydrolyzed by peptidase, releasing the hydrophilic alkylating agent melphalan. This leads to the specific release and accumulation of melphalan in aminopeptidase-positive tumor cells. Melphalan alkylates DNA at the N7 position of guanine residues, inducing intra- and inter-strand cross-links in DNA. This results in the inhibition of DNA and RNA synthesis and induces apoptosis, thereby inhibiting tumor cell proliferation. Some cancer cells overexpress peptidase. Compared to melphalan, fluoroaniline melphalan offers improved efficacy and reduced toxicity.
See also: fluoroaniline melphalan hydrochloride (note moved to). Drug Indications Fluanilide-melphalan, in combination with dexamethasone, is indicated for the treatment of adult patients with relapsed or refractory multiple myeloma who have received ≥4 lines of therapy and are resistant to at least one proteasome inhibitor, immunomodulatory agent, and anti-CD38 monoclonal antibody. The FDA withdrew this drug from the market for this indication based on data from a Phase 3 clinical trial, which showed a reduction in overall survival. Mechanism of Action Fluanilide-melphalan is a lipophilic prodrug of melphalan, making it more readily absorbed by cells. It may enter malignant cells via passive diffusion and be hydrolyzed intracellularly by aminopeptidase N. The upregulation of aminopeptidase and other hydrolytic enzymes in many malignant cells facilitates the hydrolysis of melphalan in these cells. Increased concentrations of free melphalan in malignant cells lead to rapid, irreversible DNA damage and apoptosis, thereby reducing the likelihood of resistance development. Free melphalan is an alkylating agent of a nitrogen mustard derivative. Melphalan can link an alkyl group to the N-7 position of guanine and the N-3 position of adenine, leading to the formation of a monoadduct and causing DNA fragmentation when repair enzymes attempt to correct the error. It also causes DNA cross-linking, i.e., cross-linking between the N-7 positions of one guanine and another, preventing DNA strand dissociation and thus affecting synthesis or transcription. Furthermore, melphalan can induce a variety of different mutations. Melphalan induces phosphorylation of the DNA damage marker γ-H2AX in sensitive cells within 6 hours, while melphalan-fluoroaniline induces γ-H2AX phosphorylation within 2 hours. Melphalan-fluoroaniline can also induce γ-H2AX expression in melphalan-resistant cells.

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Pharmacodynamics
Melphalan-fluoroaniline is an alkylating agent used to treat relapsed or refractory multiple myeloma. Melphalan-fluoroaniline is administered once every 28 days, thus providing a relatively long duration of action.
Patients should be informed of the risks of thrombocytopenia, neutropenia, anemia, infection, secondary malignancy, and embryo-fetal toxicity.

C24H30CL2FN3O3

498.4204

497.165

380449-51-4

Melflufen hydrochloride;380449-54-7

9935639

Typically exists as solid at room temperature

4.81

2

6

14

33

579

2

CCOC(=O)[C@H](CC1=CC=C(C=C1)F)NC(=O)[C@H](CC2=CC=C(C=C2)N(CCCl)CCCl)N

YQZNKYXGZSVEHI-VXKWHMMOSA-N

InChI=1S/C24H30Cl2FN3O3/c1-2-33-24(32)22(16-18-3-7-19(27)8-4-18)29-23(31)21(28)15-17-5-9-20(10-6-17)30(13-11-25)14-12-26/h3-10,21-22H,2,11-16,28H2,1H3,(H,29,31)/t21-,22-/m0/s1

ethyl (2S)-2-[[(2S)-2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoyl]amino]-3-(4-fluorophenyl)propanoate

J1 J-1 Prodrug J-1 MelflufenPepaxto

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)